CD1d-restricted natural killer T (NKT) cells are a distinct subset of T cells that rapidly produce an array of cytokines upon activation and play a critical role in regulating various immune responses. NKT cells are classified into two groups based on differences in T cell receptor usage. Type I NKT cells otherwise known as invariant NKT (iNKT) cells, have an invariant TCR ?-chain and are readily detectable by ?-galactosylceramide (?-GalCer)-loaded CD1d tetramers. Type II NKT cells or variant NKT cells have a more diverse TCR repertoire. While both NKT cell subsets are thought to be immunoregulatory in nature, contrasting roles of iNKT and type II NKT cells in tumor immunity have been described. The distinct functional capabilities may be attributed to distinct developmental features or unique antigen recognition. Unlike iNKT cells, little is known about thymic selection and functional regulation of type II NKT cells due to a lack of surface markers specific to this subset. In Aim 1 of this proposal, we propose to use a new animal model to identify the cell type(s) that mediate positive selection of type II NKT cells. Since altered CD1d expression has been reported under various inflammatory conditions, we also propose to investigate the effect of increased CD1d expression on the activation and function of type II NKT cells. These studies will provide information on the similarities and differences in thymic selection and peripheral activation of these two NKT cell subsets. Recent studies have found that metabolic state is extremely important for the function of conventional T cells. In particular mitochondrial function appears to play an important role in the generation of memory CD8+ T cells. Unlike conventional T cells, NKT cells exhibit an activated/memory phenotype in naive mice with several transcription factors and signaling pathways uniquely required for the development of this T cell subset. However, it is not clear whether NKT cells have distinct metabolic requirements for their development and function. To begin investigating this issue in Aim 2 of this proposal, we will utilize mice that have mitochondrial metabolic genes conditionally deleted in T cells to elucidate the role of mitochondrial metabolism in the development and function of both NKT cell subsets. In Aim 3, we propose to characterize the metabolic profiles of iNKT cells and type II NKT cells in their resting and activated states to test the hypothesis that lack of apparent iNKT memory responses may be in part due to the inability of iNKT cells to increase mitochondrial metabolism. Collectively, these studies will provide information on whether metabolic manipulation can enhance the immunotherapies that target NKT cells.